Dysferlin and animal models for dysferlinopathy

Dysferlin (DYSF) is involved in the membrane-repair process, in the intracellular vesicle system and in T-tubule development in skeletal muscle. It interacts with mitsugumin 53, annexins, caveolin-3, AHNAK, affixin, S100A10, calpain-3, tubulin and dihydropyridine receptor. Limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy (MM) are muscular dystrophies associated with recessively inherited mutations in the DYSF gene. The diseases are characterized by weakness and muscle atrophy that progress slowly and symmetrically in the proximal muscles of the limb girdles. LGMD2B and MM, which are collectively termed "dysferlinopathy", both lead to abnormalities in vesicle traffic and membrane repair at the plasma membrane in muscle fibers. SJL/J (SJL) and A/J mice are naturally occurring animal models for dysferlinopathy. Since there has been no an approach to therapy for dysferlinopathy, the immediate development of a therapeutic method for this genetic disorder is desirable. The murine models are useful in verification experiments for new therapies and they are valuable tools for identifying factors that accelerate dystrophic changes in skeletal muscle. It could be possible that the genetic or immunological background in SJL or A/J mice could modify muscle damage in experiments involving these models, because SJL and A/J mice show differences in the progress and prevalent sites of skeletal muscle lesions as well as in the gene-expression profiles of their skeletal muscle. In this review, we provide up-to-date information on the function of dysferlin, the development of possible therapies for muscle dystrophies (including dysferlinopathy) and the detection of new therapeutic targets for dysferlinopathy by means of experiments using animal models for dysferlinopathy.     

The Activity Ratio of the Cytosolic MDH/LDH and the Isoenzyme Pattern of LDH in the Peripheral Leukocytes of Dogs,Cats and Rabbits

The activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and the pattern of the isoenzymes of LDH were determined in the peripheral blood leukocytes of dogs, rabbits and cats. Rabbits had significantly higher plasma glucose concentrations than dogs or cats. Feline leukocytes showed higher LDH and lower MDH activities than canine or rabbit leukocytes. The M/L ratio, defined as the MDH activity divided by the LDH activity in cytosolic fractions, was considered to be a good indicator with which to evaluate the metabolic state in animal tissues. The M/L ratio was highest in canine and lowest in feline leukocytes. LDH-2 and LDH-3 isoenzymes were dominant in canine leukocytes. LDH-1 and LDH-2 were dominant in rabbit leukocytes, whereas LDH-5 was dominant in feline leukocytes. It was evident that there were significant differences in energy metabolism between the leukocytes of dogs, rabbits and cats.     

Characterization of monoclonal antibodies against the second-generation schizonts of Leucocytozoon caulleryi

Monoclonal antibodies (MAbs), R1 and M5, were established against the second-generation schizont of Leucocytozoon caulleryi (L. caulleryi). Both antibodies reacted to membrane and internal structure proteins of the second-generation schizont by immunofluorescence microscopy. Molecular weight of the second-generation schizont (2GS) antigen was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. At least 40 protein bands were detected in 2GS antigen by SDS-PAGE under reduced condition and ranged from 10 to 270 kDa. MAb R1 reacted to polypeptides of 150-268 kDa in 2GS antigen, whereas MAb M5 did with that of 66 kDa. Injection with a protein of 2GS antigen fractionated by affinity chromatography using MAbs R1 and M5 protected chickens against challenge with sporozoites of L. caulleryi. These results suggest that MAbs, R1 and M5, recognize 2GS antigen of L caulleryi.     

Detection and identification of the Candida species by 25S ribosomal DNA analysis in the urine of candidal cystitis

Candida species in clinical urine samples were identified directly by the newly developed method of PCR analysis on 25S ribosomal DNA (rDNA). Two dogs were referred to the Animal Medical Center, Nihon University School of Veterinary Medicine, Fujisawa, Kanagawa, Japan for the examination of chronic cystitis. Microscopic examination of urine samples from these dogs revealed yeast cells. Urine culture on Sabouraud's dextrose agar at 27 degrees C for 5 days produced white to cream colored colonies. The isolates were identifical to Candida albicans and C. parapsilosis by mycological examination, respectively. The nucleotide sequences of 25S ribosomal DNA from these urine isolates showed 99% similarity to those of a reference strain of Candida albicans or C. parapsilosis. The nucleotide sequences of 25S rDNA obtained directly from urine samples were also identical to C. albicans and C. parapsilosis, respectively. Confirming the results on the isolates cultured from the same urine samples. This PCR analysis method could be available for the direct identification of Candida species in urine samples within 2 days.     

Immunohistochemical distribution of inwardly rectifying K+ channels in the medulla oblongata of the rat

The inwardly rectifying K+ channels, Kir1.1, Kir2.3 and Kir4.1-Kir5.1, are the candidate chemosensory molecules for CO2/H+. We determined the mRNA expression and immunohistochemical localization of these channels in the medulla oblongata of the rat. RT-PCR analysis revealed mRNAs of Kir1.1, Kir2.3, Kir4.1 and Kir5.1 were detected in the medulla. The immunoreactivities for Kir1.1, Kir2.3, Kir4.1, and Kir5.1 were observed in the medulla, and immunolabeling pattern was varied by the subunit. Immunoreactivities for Kir1.1 and Kir2.3 were observed in the nerve cell bodies and glial cells both in the chemosensory areas [nucleus tractus solitarius (NTS), nucleus raphe obscurus (RO), pre-B?tzinger complex (PreB?tC)] and non-chemosensory area [hypoglossal nucleus (XII), inferior olive nucleus (IO)]. Kir4.1 immunoreactivity was observed in the glial cells and neuropil, especially in XII and IO. Kir5.1 immunoreactivity was observed in the nerve cell bodies in the XII, RO, and PreB?tC, but not in the NTS or IO. In the NTS, a dense network of varicose nerve fibers showed immunoreactivity for Kir5.1. Our findings suggest that Kir channels may not act specific to the central chemoreception, but regulate the ionic properties of cellular membranes in various neurons and glial cells.     

Repeatability and minimum harvest period of cacao (Theobroma cacao L.) in Southern Bahia

Classically, selection for superior genotypes in cacao has been based on the successive harvest records across a number of years. Little information on the minimum duration of these harvest periods is available in the literature. The repeatability coefficient (ρ) was used to estimate this period. Twenty five cacao genotypes were assayed in a randomized block design with four replications and 16-plant plots. The following yield components were studied: number of healthy fruits per plant, number of collected fruits per plant, weight of humid seeds per plant and per fruit, and percentage of diseased fruits per plant, over 5 years (1986–90). Repeatability estimates were higher than 0.84 for all components, except percentage of diseased fruits per plant (^ρ - 0.41). With such estimates, it is possible to select genotypes on the basis of only two years of successive harvests, with a determination coefficient of 90%. The advantages of applying the repeatability coefficient to the cacao breeding program are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reduction of ferric chelate caused by various wood-rot fungi

The reduction of ferric chelate caused by various wood-rot fungi was analyzed. Ferric chelate reductive activity was detected in cell-free extracts of seven wood-rot fungi:Phanerochaete chrysosporium, P. sordida YK-624,Ganoderma sp. YK-505,Coriolus versicolor, Bjerkandera adusta, Tyromyces palustris, andGloeophyllum trabeum. These fungi produced NADPH- or NADH-dependent ferric chelate reductive enzymes (or both) of different molecular weight. In the liquid culture ofP. sordida YK-624 andC. versicolor, a positive correlation was observed between extracellular MnP activity and intracellular NADPH-dependent ferric chelate reductive activity.